CRC is one of the leading causes of mortality and morbidity world-wide [1]. To the best of our knowledge, our study is the first to sequence multiple-gene panel to identify the somatic mutation pattern associated with colon cancer disease progression in a cohort of Egyptian patients to help understanding colorectal carcinogenesis process.
In the current study, the somatic mutational burden was higher in the CRC patients when compared to the other groups. The TP53, APC and ATM genes were the most frequently mutated genes in the CRC group. Matching with Cancer Genome Atlas Network, the TP53 and the APC were the most frequently mutated two genes in the CRC patients [15]. So, this finding validates the reliability of our sequencing results.
As for the TP53 which defined as the ‘guardian of the genome’; its alteration is one of the tumor hallmarks and its mutational status is associated with the progression and outcome of sporadic CRC [16]. The TP53 mutation prevalence rate in Arab population is 52.5% in comparison with 47.5% in matched Western population [17].
Our study showed that the TP53 was the 1st rank and highly mutated gene that has been detected in 73% of the CRC patients; indicating its role in the transition from an adenoma to carcinoma [18].
Eleven mutations out 13 were pathogenic with identified loss of function and have been detected only in the CRC patients. Interestingly, the most affected exon was exon4 as well as the most frequent TP53 mutations (c.121delG (58%), c.215C > G (52%)) were located in exon4. So, sequencing of TP53 exon4 could be used for CRC prediction in our population.
Matching with a recent study by Kassem et al. [19] on the Egyptian CRC patients, we found five TP53 somatic mutations; c.1024C > T, c.844C > T, c.743G > A, c.524G > A and 215C > G in our CRC group which might reveal that such variants are Egyptian specific and explain their contribution in colon cancer disease progression as a driver mutation in tumor development.
Of interest, the TP53 drug response variant (c.215C > G) [20], was detected in more than half of our CRC group. Also, it has been recently observed in 17% of the Egyptian breast cancer patients [21]. Thus, this variant might serve as an efficient predictive marker for chemotherapy response in the Egyptian cancer patients.
Our pathway analysis revealed that the P53 signaling pathway was inactivated in 72% of the CRC patient due to either loss of the TP53 wild type or oncogenic gain of the TP53 mutant. Several new therapies specifically target p53-mutant cells, while others correct the p53 mutations directly or restore the integrity of the p53 pathway [22]. Therefore, the deficient p53 signaling pathway in our CRC group may arise as an attractive therapeutic target.
Mutation of the APC gene, a multi-functional tumor-suppressor gene, is an early event in the development of CRC and result in activation of Wnt/β-catenin signaling pathway [23]. Mutant APC, Axin2 along with AMER1 (APC-recruitment protein) disrupt the formation of β-catenin destruction complex leading to stabilization and accumulation of β-catenin protein which in turn induces overactivation Wnt/β-catenin signaling and promote the proliferation, invasion and metastasis of cancerous cells [24, 25].
We have found that the APC gene was the 2nd rank highly mutated gene in 69% of the CRC patients. Fifteen mutations out of 17 were pathogenic somatic mutations with identified loss of function detected only in the CRC group. Interestingly, exon 16 was the most affected exon and it was found to harbor 14 out of 15 detected mutations in the CRC group. Moreover, the most frequently detected mutation in the APC gene that found to be associated with disease progression (c.3754delT (65%)) was also in exon 16. So, sequencing of exon 16 could be used as genetic test assay for CRC diagnosis. Of interest, most of our identified somatic mutations were located in the β-catenin and downregualtion site.
According to COSMIC database, we detected two APC variants (c.4588G > T (2%) and c.5288delA (2%)) that were previously reported to be found respectively in pancreatic cancer and hepatocellular carcinoma. Interestingly, this is the first study to report the presence of such variants in the CRC and to report their involvement in the colorectal carcinogenesis process. However, further studies are needed to validate our findings.
We also detected three AXIN2 mutations in 27% of the CRC patients. Two out of them (c.2347G > T & c.1975C > T) were reported in 2% and 5% respectively in the CRC group only. It was previously reported by Imielinski et al. and George et al. that these two mutations are associated with small cell lung cancer [26, 27]. So, this study is the first to report their association with CRC.
In this study and according to the IPA software, we found that Wnt/βcatenin pathway was upregulated in 73% of the CRC patients; revealing that Wnt/βcatenin pathway plays a major role in sporadic colorectal carcinogenesis and therefore it is an attractive target for therapeutic intervention [28].
Somatic mutations of the ATM gene, as a DNA repair gene, occur in many tumor types including colorectal cancer. In the colorectal cancer, loss of ATM protein expression is associated with worse prognosis [29]. So, we are in need for such targeted sequencing studies to help monitoring the prognosis in Egyptian CRC patients.
Our data revealed that the ATM has been mutated in 44% of the CRC patients. We detected 9 somatic mutations in the ATM gene, 6 out of them were detected only in the CRC group. The identified ATM mutations were previously reported to be associated with CRC (Zehir et al., 2017). Also, we detected two other ATM polymorphisms (c.9007A > G & c.8138G > A), however they were reported to be associated with NHL lymphoma [30].
Moreover, this study showed that the ATM signaling pathway was downregulated in 72% of the CRC patients; revealing the critical role of the defective DNA repair mechanism in colorectal carcinogenesis process [31].
Nowadays, novel therapies have been developed to selectively target patients with ATM-deficient cancers. Those therapies induce synthetic lethality due to lacking efficient repair mechanism such as platinum drugs [32]. Thus, the ATM mutational status could be used to help in the clinical decision-making for those patients along with the development of specific targeted strategies [33]. Thus, it is important to conduct targeted sequencing studies on the Egyptian CRC patients to evaluate drug efficacy and treatment protocols.
Matching with two studies that reported the association of SMAD4 mutations with the CRC, we detected four somatic mutations (c.692delG, c.1064A > G, c.1081C > T & c.1088G > A) only in the CRC group [34]. The SMAD4 gene acts as an intracellular mediator of TGF-β superfamily signals. TGF-β/SMAD4 signaling maintains DNA damage response (DDR) and DNA damage repair [35]. In this study, the TGF beta pathway was downregulated in 36% of the CRC patients. It was suggested that loss or downregulation of the SMAD4 promotes malignant progression via acquiring resistance to TGF- β superfamily growth inhibition [36]. Moreover, its loss shifts TGF-β signaling pathway to a tumor promoter instead of a tumor suppressor [37]. Our findings revealed that the SMAD4 mutations had prominent role in colorectal carcinogenesis. Isaksson-Mettavainio et al. reported that loss of the SMAD4 occurs in the CRC in frequencies ranging from 9 to 67% [38]. Moreover, the SMAD4 loss was also associated with worse clinical outcome and resistance to fluoropyrimidine-based chemotherapy [39]; implicating its use as a prognostic marker in the CRC patients [40]. Thus, we propose that the Egyptian CRC patients carrying SMAD4 mutations may not benefit from fluoropyrimidine-based treatment.
Functional loss of the putative tumor suppressor EP300 gene has been previously observed in gastric, breast, pancreatic, and colorectal cancers. Also, Gayther et al reported a great relevance of the EP300 loss in the colorectal carcinogenesis [41]. Our study found that the EP300 gene harbored 3 somatic mutations. Out of them, one frameshift mutation (c.832delA) was found to be associated with CRC [42], while the other ones, the splice donor & missense mutations (c.1058G > A &c.3671 + 1G > A), were respectively detected in breast and gastric cancers [43, 44]. Moreover, Huh et al. reported that p300 overexpression was an indicator of good prognosis in the CRC patients [45]. Therefore, the identified somatic mutations in the EP300 gene might serve as predictor of bad prognosis in the Egyptian CRC patients.
One of the most frequently detected somatic mutations in the CRC is in the tumor suppressor FBXW7 gene. Loss of the FBXW7 was reported to promote epithelial-mesenchymal transition (EMT) and metastasis in the CRC cells [46]. The present study reported two somatic mutations with functional loss in FBXW7 gene (c.2001delG & c.4900A > G). These mutations were previously reported to be found mainly in the CRC patients [47, 34]. Moreover, a recent study reported the presence of an association between the FBXW7 mutations and resistance to anti-epidermal growth factor receptor (EGFR) immunotherapy treatment that commonly used to manage metastatic CRC [48]. So, identifying the mutational status of the FBXW7 gene in Egyptian CRC patients may serve as a good diagnostic biomarker to determine the appropriate individualized therapy [49].
The functional loss of the tumor suppressor ARIDA1 gene has been previously reported as a frequent event in the colorectal carcinogenesis [50]. In agreement with a previous study by Erfani et al. who reported a relatively high mutation rate of the ARID1A in the CRC ranging from 10% up to 40%, we also reported a high mutation rate of the ARIDA1 around 29% in our CRC group [51]. Matching with a previous study which showed that the ARIDA1 mutations were more likely to be frameshift or nonsense, we detected 5 framshift mutations in the ARIDA1 [50]. Additionally, four mutations out of them were detected only in the CRC group. Thus, our study showed the prominent role of the ARIDA1 gene in the colorectal carcinogenesis.
Matching with several previous studies [52–54], we detected many gain of function somatic mutations that have been previously reported to be commonly detected in the CRC; BRAF (c.1781A > G, c.1799T > A), KRAS (c.35G > T, 38G > A), NRAS (c.35G > T) &PIK3CA (c.3140A > G), ERBB2 (c.922G > A & c.2690G > A). Additionally, other reports revealed that these mutations contribute to the acquired resistance to the anti-EGFR therapy [55, 56]. Thus, genetic testing of those genes provides beneficial information that help in the clinical management of the CRC patients.
Regarding the common somatic mutations detected in all the studied groups; we have found that nine out of twenty four somatic mutations were the most frequent (c.1310delA in ACVR2A), (c.640delT in APC), (c.5557G > A in ATM), (c.677delG in IGF2), (c.1621A > C in KIT), (c.1173A > G in PIK3CA), (c.2071G > A in RET), & (c.121delG, c.215C > G in TP53). The frequency of those mutations increased from the colitis to finally the CRC, so they could be used as predictors for disease progression. Most of the above identified somatic mutations were observed mostly with the CRC [47], except for (c.640delT in ATM), (c.1621A > C in KIT), (c.2071G > A in RET) & (c.121delG in TP53) which were observed respectively in breast [57], soft tissues [58] & head and neck cancers [59]. This is the first study to report their association with the colorectal carcinogenesis in Egyptian CRC patients.